The genome of influenza A and B viruses is composed of eight single-strand RNA segments of negative polarity, two of which encode envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Replication of influenza virus is initiated by the binding of the viral HA proteins on the virion surface to cellular sialic acid containing receptors. After binding to the receptors, virions are taken into the host cells by endocytosis. The acidic environment in the late endosome triggers HA conformational changes, initiating fusion between the viral envelope and the endosomal membrane, and activates the M2 ion channel, resulting in proton influx into the virion interior. Exposure of the virion interior to low pH is thought to disrupt acid-labile interactions between the M1 protein and ribonucleoprotein complex (RNP), culminating in the release of RNP into the cytoplasm. The RNP is then transported to the nucleus, where viral mRNA and the viral genome are synthesized. mRNA enters the cytoplasm and viral proteins are synthesized. Nucleoprotein (NP) enters the nucleus and encapsidates newly synthesized vRNA and, together with the three polymerase subunit proteins (PA, PB1, PB2), forms RNP. In the presence of M1 and NS2 proteins, RNP is exported out of the nucleus. The three plasma membrane-associated proteins (HA, NA and M2) and RNP interact and form new virions by budding. NA is responsible for viral release from infected cells by removing sialic acids from cellular glycoconjugates and viral glycoproteins (Lamb et al., 2000).
Type A viruses are divided into subtypes based on HA (H1–H15) and NA (N1–N9) antigenicities. In cells infected with two different type A viruses, intratypic reassortants possessing various combinations of gene segments are produced (Wright et al., 2000). However, intertypic reassortants between type A and B viruses have not been detected in nature, although both viruses are cocirculating in human populations.
Investigators have attempted to generate reassortants between type A and B viruses in the laboratory without success (Kaverin et al, 1983; Mikheera et al., 1982; Tobita et al., 1983). Muster et al. (1991) generated a mutant type A virus containing a segment in which the noncoding regions of a NA segment were replaced with those of the nonstructural (NS) gene of type B virus. Although the mutant virus replicated more slowly and achieved lower titers than wild-type virus, the generation of such a virus suggested that the noncoding regions of the type B NS segment were compatible with influenza virus type A components at the level of RNA transcription and replication. By contrast, an RNA segment possessing a foreign coding segment flanked by the 3′ and 5′ noncoding regions of an influenza A viral RNA segment, was not stably maintained in virions after repeated passage (Luytjes et al., 1989). Muster et al. (1991) also disclose that the mutant virus was attenuated in mice, and that animals infected with the mutant virus were resistant to challenge with the wild-type virus.
What is needed is a method to identify influenza virus sequences for incorporation and/or maintenance of linked sequence during influenza virus replication.